CN116354917A - 一种金沙江石斛内生真菌色原酮类化合物dwt001及其制备和应用 - Google Patents
一种金沙江石斛内生真菌色原酮类化合物dwt001及其制备和应用 Download PDFInfo
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Abstract
本发明涉及天然产物制备技术领域,公开了一种金沙江石斛内生真菌色原酮类化合物DWT001及其制备和应用,其制备方法包括如下步骤:步骤一、将石斛内生菌培养、发酵得发酵液;步骤二、将发酵液减压浓缩后萃取,并对萃取液进行浓缩得浸膏;步骤三、将浸膏梯度洗脱、显色合并后,分为A~F6个粗组分;步骤四、将组分D经中压液相色谱梯度洗脱,得到9个亚组分D1~D9;步骤五、将组分D3经正相硅胶柱色谱梯度洗脱,得到8个亚组分D3‑1~D3‑8;步骤六、将组分D3‑6经制备型HPLC分离,得到化合物DWT001。该化合物为新的chromone类化合物;经过抗人类病原菌活性的检测,化合物DWT001表现出对大肠埃希菌E.coli和白色念珠菌C.albicans较好的抑制活性。
Description
技术领域
本发明涉及天然产物制备技术领域,具体涉及一种金沙江石斛内生真菌色原酮类化合物DWT001及其制备和应用。
背景技术
金沙江石斛(Dendrobium wangliangii G.W.Hu,C.L.Long et X.H.Jin),是我国兰科特有种类,为金沙江干热河谷云南段特有类群,在药用及观赏方面有着潜在价值。但由于分布区域狭窄、气候异常以及认为采集等诸多因素的影响,金沙江石斛目前濒临灭绝,亟待保护。成熟的兰科植物种子极其微小且没有储存营养的胚乳,这使得兰科植物在长期进化中,形成了独特的与内生真菌的共萌发出芽的方式。胶膜属真菌Tullasnella sp.就是其中一种,目前有研究发现,该属内生真菌能对不同种类的石斛种子产生促萌发作用。
内生真菌能产生种类丰富且具有良好生物活性的代谢产物,但在内生真菌Tullasnella sp.与宿主植物金沙江石斛种子共萌发过程中哪些化合物能促进种子萌发,目前还尚不清楚。所以对金沙江石斛内生真菌Tullasnella sp.的化学成分及其生物活性的研究,能够为金沙江石斛的保护保育工作提供化学基础,同时也能挖掘有良好活性的新颖化合物。
发明内容
本发明意在提供一种金沙江石斛内生真菌色原酮类化合物DWT001及其制备和应用,以实现石斛种子的萌发促进并开发该化合物的生物活性。
为达到上述目的,本发明采用如下技术方案:一种金沙江石斛内生真菌色原酮类化合物DWT001,其特征在于:化合物结构如式(I)所示:
另一方面,本技术方案提供一种金沙江石斛内生真菌色原酮类化合物DWT001的制备方法,包括如下步骤:
步骤一、将石斛内生菌培养、发酵得发酵液;
步骤二、将发酵液减压浓缩后萃取,并对萃取液进行浓缩得浸膏;
步骤三、将浸膏梯度洗脱、显色合并后,分为A~F 6个粗组分;
步骤四、将组分D经中压液相色谱梯度洗脱,得到9个亚组分D1~D9;
步骤五、将组分D3经正相硅胶柱色谱梯度洗脱,得到8个亚组分D3-1~D3-8;
步骤六、将组分D3-6经制备型HPLC分离,得到化合物DWT001。
再一方面,本技术方案还提供一种金沙江石斛内生真菌色原酮类化合物DWT001在制备抑菌剂中的应用。
优选的,作为一种改进,步骤一中,培养条件为在PDB液体培养基里25℃恒温培养5d。
优选的,作为一种改进,步骤一中,发酵培养基组成为:5%葡萄糖、0.15%蛋白胨、0.5%酵母、0.05% KH2PO4、0.05% MgSO4、余量为水;发酵条件为24-28℃、140-180rpm条件下液体发酵一个月。
优选的,作为一种改进,步骤二中,萃取剂为乙酸乙酯,萃取次数为3次。
优选的,作为一种改进,步骤三中,采用200-300目正相硅胶柱色谱洗脱,洗脱时氯仿与甲醇的体积比依次为1:0、50:1、30:1、15:1、10:1、5:1、3:1、2:1、1:1。
优选的,作为一种改进,步骤四中,洗脱液为甲醇-水,洗脱时甲醇与水的体积比依次为20:80、40:60、60:40、80:20、100:0。
优选的,作为一种改进,步骤五中,洗脱液为石油醚-丙酮体系,洗脱时石油醚与丙酮的体积比依次为8:1、6:1、4:1、2:1、1:1、0:1。
优选的,作为一种改进,步骤六中,分离液为甲醇-水,甲醇与水的体积比为40:60-44:56,流速4mL/min,分离时间20min。
本方案的原理及优点是:实际应用时,本技术方案中,从石斛内生真菌Tullasnella sp.的发酵液中通过梯度洗脱剂薄层色谱分析得到不同的组分,从特定组分中分离得到1个新的化合物(DWT001),经核磁共振及质谱鉴定,DWT001为新的chromone类化合物。经过抗人类病原菌活性的检测,化合物DWT001表现出对大肠埃希菌E.coli和白色念珠菌C.albicans较好的抑制活性。
附图说明
图1为本发明金沙江石斛内生真菌Tullasnella sp.平板图。
图2为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001氢谱。
图3为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001碳谱。
图4为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001 HSQC图谱。
图5为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001 HMBC图谱。
图6为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001 1H-1HCOSY图谱。
图7为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001 ROESY图谱。
图8为本发明实施例中金沙江石斛内生真菌色原酮类化合物DWT001 MS图谱。
具体实施方式
下面通过具体实施方式进一步详细说明,但本发明的实施方式不限于此。若未特别指明,下述实施方式所用的技术手段为本领域技术人员所熟知的常规手段;所用的实验方法均为常规方法;所用的材料、试剂等,均可从商业途径得到。
一种金沙江石斛内生真菌色原酮类化合物DWT001,化合物结构如式(I)所示:
一种金沙江石斛内生真菌色原酮类化合物DWT001的制备方法,包括如下步骤:
步骤一、发酵液的制备:将石斛内生真菌Tullasnella sp.在PDB液体培养基里25℃恒温培养5天。然后将培养基倒入含有液体培养基的发酵罐中,在25℃和160rpm条件下液体发酵一个月,最后获得发酵液25L(液体培养基组成:5%葡萄糖,0.15%蛋白胨,0.5%酵母,0.05% KH2PO4和0.05% MgSO4,余量为水)。其中,金沙江石斛内生真菌Tullasnellasp.为云南大学生态与环境学院提供。
步骤二、化合物DWT001的制备:将25L发酵液减压浓缩至5L,用4L乙酸乙酯反复萃取三次,乙酸乙酯萃取液浓缩后获得浸膏136g。采用200-300目正相硅胶柱色谱,用氯仿-甲醇为洗脱液(1:0-0:1,v/v)梯度洗脱,洗脱时氯仿与甲醇的体积比依次为1:0、50:1、30:1、15:1、10:1、5:1、3:1、2:1、1:1,通过薄层色谱(TLC)显色合并,将粗浸膏分为6个粗组分(A~F)。
组分D(9.3g)经中压液相色谱(甲醇-水,20:80-100:0,v/v)梯度洗脱,洗脱时甲醇与水的体积比依次为20:80、40:60、60:40、80:20、100:0,得到9个亚组分D1~D9。
组分D3(389.0mg)经正相硅胶柱色谱石油醚-丙酮体系(8:1-0:1,v/v)梯度洗脱,洗脱时石油醚与丙酮的体积比依次为8:1、6:1、4:1、2:1、1:1、0:1,得到8个亚组分D3-1~D3-8。
组分D3-6经制备型HPLC(甲醇-水,40:60-44:56,20min,4mL/min)分离,得到化合物DWT001(3.3mg,tR=14.5min)。
一种金沙江石斛内生真菌色原酮类化合物DWT001在制备抑菌剂中的应用。
实验例一金沙江石斛内生真菌色原酮类化合物DWT001的鉴定
从石斛内生真菌Tullasnella sp.的发酵液中分离得到1个新化合物(DWT001),经经核磁共振氢谱、碳谱、HSQC谱、HMBC谱等波谱数据及质谱数据鉴定,DWT001为新的chromone类化合物(图2-图8)。
实验例二金沙江石斛内生真菌色原酮类化合物DWT001的抑菌作用研究
实验方法:通过微量肉汤稀释法,检测化合物对大肠埃希菌Escherichia coli(ATCC 25922)和白色念珠菌Candida albicans(ATCC 10231)的抑制作用。将上述两个病原菌接种于PDB液体培养基,在全温培养摇床过夜培养(37℃,200rpm)。化合物用DMSO溶解浓度为1mg/mL,然后用超纯水分别调为16μg/mL,32μg/mL,64μg/mL,128μg/mL,256μg/mL,512μg/mL,并对应地将DMSO也用超纯水调为同浓度溶液。
设置实验组为不同浓度100μL化合物+100μL菌液,按浓度由高到低顺序加入孔中,3复孔;阴性对照1为200μL MH或PDB培养基,2复孔;阴性对照2为100μL化合物+100μL培养基,2复孔;阳性对照为100μL氯霉素(E.coli)或氟康唑(C.albicans)+100μL菌液,2复孔;空白对照为200μL菌液,2复孔。将96孔板放入高通量生长曲线分析仪中震荡培养24小时,观察结果。观察化合物浓度为8μg/mL,16μg/mL,32μg/mL,64μg/mL,128μg/mL,256μg/mL和512μg/mL时的菌液生长情况。空白对照孔显示浑浊,阴性对照显示清亮,质控在规定范围内,以肉眼观察无细菌生长的最低浓度即为化合物的最小抑菌浓度(MIC)值。
对化合物1进行了抗人类病原菌活性的检测,其表现了较好的抗菌活性,结果如表1所示。
表1化合物1抗病原菌活性(MIC,μg/mL)
以上所述的仅是本发明的实施例,方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出,对于本领域的技术人员来说,在不脱离本发明技术方案的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (10)
2.根据权利要求1所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备方法,其特征在于,包括如下步骤:
步骤一、将石斛内生菌培养、发酵得发酵液;
步骤二、将发酵液减压浓缩后萃取,并对萃取液进行浓缩得浸膏;
步骤三、将浸膏梯度洗脱、显色合并后,分为A~F 6个粗组分;
步骤四、将组分D经中压液相色谱梯度洗脱,得到9个亚组分D1~D9;
步骤五、将组分D3经正相硅胶柱色谱梯度洗脱,得到8个亚组分D3-1~D3-8;
步骤六、将组分D3-6经制备型HPLC分离,得到化合物DWT001。
3.根据权利要求2所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤一中,培养条件为在PDB液体培养基里25℃恒温培养5d。
4.根据权利要求3所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤一中,发酵培养基组成为:5%葡萄糖、0.15%蛋白胨、0.5%酵母、0.05%KH2PO4、0.05%MgSO4、余量为水;发酵条件为24-28℃、140-180rpm条件下液体发酵一个月。
5.根据权利要求4所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤二中,萃取剂为乙酸乙酯,萃取次数为3次。
6.根据权利要求5所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤三中,采用200-300目正相硅胶柱色谱洗脱,洗脱液为氯仿-甲醇溶液,洗脱时氯仿与甲醇的体积比依次为1:0、50:1、30:1、15:1、10:1、5:1、3:1、2:1、1:1。
7.根据权利要求6所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤四中,洗脱液为甲醇-水,洗脱时甲醇与水的体积比依次为20:80、40:60、60:40、80:20、100:0。
8.根据权利要求7所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤五中,洗脱液为石油醚-丙酮体系,洗脱时石油醚与丙酮的体积比依次为8:1、6:1、4:1、2:1、1:1、0:1。
9.根据权利要求8所述的一种金沙江石斛内生真菌色原酮类化合物DWT001的制备和应用,其特征在于:步骤六中,分离液为甲醇-水,甲醇与水的体积比为40:60-44:56,流速4mL/min,分离时间20min。
10.一种金沙江石斛内生真菌色原酮类化合物DWT001在制备抑菌剂中的应用。
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